Processive myosins transport intracellular cargo, such as organelles and mRNA, along actin tracks for several micrometers before dissociating (i.e. processivity). We will probe the basis for processive motion in a double-headed (class V) and a single-headed (class IXb) myosin motor, and investigate how actin- and microtubule-based motors interact with each other. Mutant myosins will be expressed in the baculovirus/insect cell expression system. A key new technique is a TIRF (total internal reflectance fluorescence)-microscopy assay which will be used to directly assess processive run lengths and velocities of single-myosin molecules. Other assays include in vitro motility, kinetic analysis, and hydrodynamic techniques. In Aim #1 we seek to convert a non-processive class V myosin (Drosophila) into a processive motor (murine myosin V). Many differences between these two class V myosins are clustered in key regions in the motor domain. The neck region has been proposed to mediate coordination between the two-heads during processive motion. We will alter its compliance and assess the impact on processive movement. Lastly, we will test the idea that a monomer-dimer equilibrium regulates processivity in yeast class V myosins. In Aim #2 we will determine if the unique large insertion in loop 2 and the N-terminal extension is necessary for single-headed myosin IXb to achieve processivity. Actin-bound myosin IX will be visualized by electron cryomicroscopy to identify the structural role of the two unique insertions, and the mode of myosin IX binding to actin in different nucleotide states (consortium with Hanein). In Aim #3 we will test the hypothesis that myosin V and kinesin interact directly and co-operate to facilitate transport of cargo from microtubules to actin tracks. Processive motion will be followed at the single molecule level using TIRF-microscopy. Structural approaches (collaboration with K. Taylor) will be used to gain insight into how the motors interact in the absence or presence of their tracks. Mutations in myosin V lead to Griscelli syndrome (hypopigmentation and neurological impairment). Myosin IX plays a role in cell-signaling, because the GAP domain in its tail (its "cargo") is believed to regulate Rho-dependent remodeling of the cytoskeleton in motile cells and in the dynamic arrays of actin in synapses. A number of human diseases result from disruption of cargo transport along actin or microtubules, or from disease proteins acting as cargo for these transport proteins. A better understanding of the mechanism by which molecular motors move along their track is critical to further understand the basis of these diseases.